Identification of noncollagenous sites encoding specific interactions and quaternary assembly of alpha 3 alpha 4 alpha 5(IV) collagen: implications for Alport gene therapy.
Defective assembly of alpha 3 alpha 4 alpha 5(IV) collagen in the glomerular basement membrane causes Alport syndrome, a hereditary glomerulonephritis progressing to end-stage kidney failure. Assembly of collagen IV chains into heterotrimeric molecules and networks is driven by their noncollagenous (NC1) domains, but the sites encoding the specificity of these interactions are not known. To identify the sites directing quaternary assembly of alpha 3 alpha 4 alpha 5(IV) collagen, correctly folded NC1 chimeras were produced, and their interactions with other NC1 monomers were evaluated. All alpha1/alpha 5 chimeras containing alpha 5 NC1 residues 188-227 replicated the ability of alpha 5 NC1 to bind to alpha3NC1 and co-assemble into NC1 hexamers. Conversely, substitution of alpha 5 NC1 residues 188-227 by alpha1NC1 abolished these quaternary interactions. The amino-terminal 58 residues of alpha3NC1 encoded binding to alpha 5 NC1, but this interaction was not sufficient for hexamer co-assembly. Because alpha 5 NC1 residues 188-227 are necessary and sufficient for assembly into alpha 3 alpha 4 alpha 5 NC1 hexamers, whereas the immunodominant alloantigenic sites of alpha 5 NC1 do not encode specific quaternary interactions, the findings provide a basis for the rational design of less immunogenic alpha 5(IV) collagen constructs for the gene therapy of X-linked Alport patients.
Kang
Jeong Suk
JS
Department of Medicine, Division of Nephrology, Vanderbilt University, Nashville, Tennessee 37232, USA.
Colon
Selene
S
Hellmark
Thomas
T
Sado
Yoshikazu
Y
Hudson
Billy G
BG
Borza
Dorin-Bogdan
DB
D000595:Amino Acid Sequence; D000941:Antigens / Q000737:chemistry; D001665:Binding Sites; D024141:Collagen Type IV / Q000737:chemistry* / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009394:Nephritis, Hereditary / Q000235:genetics* / Q000628:therapy; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D000941:Antigens; D024141:Collagen Type IV; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins